A Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detection of HCV Genotypes 1b and 2a in China.

Abstract

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was used to detect hepatitis C virus (HCV) lb and 2a (the major genotypes in China). First, clinical samples were collected, and the genotypes and viral load determined. Second, RT-LAMP primers of HCV lb and 2a were designed according to the 5' untranslated region of the HCV. Third, the specificity of RT-LAMP was tested by non-lb and non-2a HCV viruses, and the products analyzed by endonuclease. Fourth, the limit of detection was tested using diluted samples, and the results determined by calcein/Mn(2+) -dependent visual methods. Finally, the consistency of RT-LAMP and real-time fluorescent polymerase chain reaction (PCR) was compared to evaluate the clinical practicality. Results showed that RT-LAMP primers had good specificity because there was no cross-reactivity and the target sequences were identified correctly by endonuclease. The limit of detection was 100 IU/mL and calcein/Mn(Z+)-dependent visual methods were easier to use compared with electrophoresis. After analyses of all amplification results by statistical software; we found no significant difference between RT-LAMP and real-time fluorescent PCR (P> 0. 05). In conclusion, RT-LAMP requires only simple apparatus and simple operation, which is very helpful in primary-health-care institutions.